The proposed research project addresses basic and translational aspects of the function of thrombin as an anticoagulant, which has received far less attention than the procoagulant and prothrombotic roles mediated by cleavage of fibrinogen and the platelet receptor PAR1, respectively. Specifically, the project deals with the molecular mechanism of protein C activation by thrombin and its connection with the mechanism of prothrombin activation, as well as with the possibility of turning thrombin into an exclusive activator of protein C for clinical applications. We will address the following basic questions: What is the mechanism of protein C activation by thrombin? Is this mechanism related to that of prothrombin activation? Can thrombin be converted into an exclusive activator of protein C by selectively abrogating activity toward fibrinogen and PAR1? The project builds upon recent exciting developments and consists of the following specific aims: 1. Elucidate the molecular mechanism of prethrombin-2 activation and auto-activation; 2. Elucidate the molecular mechanism of protein C activation by thrombin; 3. Convert thrombin into an exclusive activator of protein C. In specific aim 1, we will build on a recent breakthrough structure of the thrombin precursor prethrombin-2 in the free form where R15 at the site of cleavage in the activation domain is buried inside the protein, in ionic interaction with E14e, D14l and E18. This observation is unique among existing structures of trypsin-like zymogens and bears on the molecular mechanism of prothrombin activation. Remarkably, mutation of E14e, D14l and E18 to Ala generates a prethrombin-2 mutant that auto-activates to thrombin without the need for the snake venom ecarin or the physiological prothrombinase complex. We will elucidate the mechanism and factors that control this remarkable property of prethrombin-2 in the context of prothrombin activation using site-directed mutagenesis, kinetics and X-ray structural biology. In specific aim 2, we will exploit the striking sequence similarity between protein C and prethrombin-2 in the activation domain, with E14e, D14l and E18 around the site of cleavage at R15 in prethrombin-2 replaced by E160, D167 and D172 around the site of cleavage at R169 in protein C. We will test the hypothesis that R169 of protein C is not exposed to solvent and that the action of thrombomodulin is to induce exposure of R169 to enable thrombin cleavage. We also hypothesize that mutations of protein C in the activation domain result in constructs with enhanced activation by thrombin or even capable of auto-activation. In specific aim 3, we will use a combination of existing mutations that produce a shift in substrate specificity in favor of protein C to generate a new class of therapeutically relevant thrombin mutants that have completely lost activity toward the procoagulant substrate fibrinogen and the prothrombotic substrate PAR1, but retain activity toward the anticoagulant substrate protein C in the presence of the cofactor thrombomodulin. PUBLIC HEALTH RELEVANCE: Recent statistics indicate that cardiovascular disease and its thrombotic complications will remain the leading cause of death and disability and will represent a major burden to productivity in the US and worldwide well into the year 2020. Because of its involvement in thrombotic deaths, thrombin remains a major target of antithrombotic and anticoagulant therapies. Progress in the understanding of the anticoagulant properties of thrombin and how they can be exploited to engineer a new class of anticoagulants has the potential to influence the life-style and life expectancy of millions of people in the US and worldwide.